A. Field of the Invention
The present invention relates to the spatial and temporal control of exogenous gene expression in genetically engineered cells and organisms. In particular, the invention relates to the use of heat-inducible promoters such as the promoter of heat shock genes to control the expression of exogenous genes. More particularly, the invention relates to the use of focused ultrasound to heat cells that contain therapeutic genes under the control of a heat shock promoter, thereby inducing the expression of the therapeutic gene.
B. Description of Related Art
Disorders caused by a malfunctioning gene can-be treated by stably transferring an exogenous functional gene into a host cell, so that the gene product of that gene is produced in the host cell. Gene transfer may also be used to express in a host cell exogenous nucleic acids that kill the host cell, or that encode gene products that alter the phenotype of the host cell and/or the metabolic state of surrounding cells, or that suppress the expression of selected genes in the host cell. Human diseases are amenable to treatment by this approach, particularly those diseases where the defect is with a single gene. For discussions on the application of gene therapy towards the treatment of genetic as well as acquired diseases See Miller, A. D. (1992) Nature 357:455–460, and Mulligan, R. C. (1993) Science 260:926–932, both incorporated herein by reference.
In many instances, it is desirable to express genetically engineered genes only in certain tissues, and/or at will only at certain times, and/or only to a certain degree. However, current gene transfer and exogenous gene expression protocols do not provide adequate means of simultaneously controlling which cells in a heterogeneous population are transformed, and when, where and to what degree the transferred genes are expressed.
One approach for the control of exogenous gene expression that has received a great deal of attention is to transform host cells with a gene that is under the control of an inducible promoter, and then to switch the transferred gene on and off at will by activating the inducible promoter. Inducible promoters include: the metallothionine IIA promoter, the lacZ, tac, and trp promoters, the phage T7 promoter/T7 RNA polymerase, the Candida albicans MAL2 gene promoter. Some promoters are heat inducible: e.g., the lambda PL promoter with a C1857 repressor, heat shock protein promoters.
Heat shock proteins (“hsps”) are a ubiquitous class of proteins produced in response to stress, notably heat stress, as well as a variety of other external agents. All cells that have so far been tested contain-hsps, and many different hsps have been identified in a wide range of organisms. Many hsps are designated according to their molecular mass, (e.g. hsp70 refers to a 70 kDalton hsp; hsp56, hsp28). Additional examples of hsps include ubiquitin, crystallin, rapamycin, P-glycoprotein, and others. The following articles describe the properties of heat shock genes and promoters: Yost et al., (1990) TIG, 6:222–226. RNA metabolism: strategies for regulation in the heat shock response. Pennier, (1994) Biochemie, 76:737–747. Translational control during heat shock; Minowada and Welch, (1995) “The Clinical implications of the stress response”, J. Clin. Invest. 95:3–12; Lis and Wu, (1993) Cell 74:1–4. Protein traffic on the heat shock promoter: parking, stalling, and trucking along; Holbrook and Udelsman, “Heat shock protein gene expression in response to physiological stress and aging,” in THE BIOLOGY OF HEAT SHOCK PROTEINS AND MOLECULAR CHAPERONES” (Morimoto et al., (1994) Editors, Plainview, N.Y.: Cold Spring Harbor Laboratory Press, pp. 577–593); Macario, (1995), “Heat-shock proteins and molecular chaperones: implications for pathogenesis, diagnostics, and therapeutics,” Int. J. Chem. Lab Res. 25:59–70.
Hsps participate in and influence a large variety of cellular effects, including the assembly of newly formed polypeptides (some HSPs function as chaperones), signalling functions (e.g. response to steroid hormones), protein excretion, DNA and RNA synthesis (see below). The synthesis of proteins during heat shock is generally inhibited during stress, except for the synthesis of the hsps.
Heat shock (and other forms of stress) result in the almost immediate transcriptional activation of heat shock genes. The heat shock response is quite dramatic. The heat shock messages appear in the cytoplasm generally within minutes, and the translation of message is carried out with a very high efficiency. For example, in Drosophila cells, hsp genes are induced within just four minutes after a temperature elevation of 4 to 9° C. Within one hour, there are several thousand transcripts per cell. These transcripts are actively translated into hsp while at-the same time, the transcription of previously active genes is severely repressed. Miller and Ziskin (1990) Ultrasound Med. Biol. 15:707–22 reported that short exposures to sharply elevated temperature result in a protective effect against further thermal insult, and that the generation of heat shock proteins by cells coincides with the onset of such “thermal protection. “The level of synthesis of hsp70 in cells during heat shock appears to be linearly related to their thermotolerance. Li, G. C. (1985) Int. J. Radiat. Oncol. Biol. Phys. 11:165–177. Two human hsp70 proteins have been described-hsp70A (Wu, B., et al. (1985) Mol. Cell. Biol. 5:330–341; Hunt, C., and Morimoto, R. I. (1985) Proc. Natl. Acad. Sci. USA 82.: 6455–6459) and hsp70B (Schiller, P., et al. (1988) J. Mol. Biol. 203:97–105). For a review of hsps, see, e.g., Morimoto et al., eds., Stress Proteins in Biology and Medicine (1990) Cold Spring Harbor Press; Hightower, L. E. (1991) Cell 66:191–197.; Craig, E. A., and Gross, C. A. (1991) Trends Bioch. Sci. 16:135.
The heat shock genes of many organisms have been mapped and sequenced. Heat shock genes are scattered at various chromosomal locations. A remarkable feature of these genes is the general absence of any intervening sequences.
Heat shock promoters from different sources have been have been isolated, sequenced and used to express a variety of genes. For example, Dreano, M., et al. (1986) Gene 49:1–8, describe the use of the human hsp70B promoter, as well as a Drosophila hsp70 promoter, to direct the heat regulated synthesis of human growth hormone, chicken lysozyme and a human influenza hemagglutinin. EPA Publication No. 336,523 (Dreano et al., published 11 Oct. 1989) describes the in vivo expression of human growth hormone using a human hsp70 promoter. PCT Publication No. WO 87/00861 (Bromley et al., published 12 Feb. 1987) describes the use of human and Drosophila hsp promoters having 5′-untranslated region variants. EPA Publication No. 118,393 (Bromley et al., published 12 Sep. 1984) and PCT Publication No. WO 87/05935 (Bromley et al., published 8 Oct. 1987) describe the expression of E. coli beta-galactosidase and human influenza hemagglutinin, using a Drosophila hsp70 promoter. See also U.S. Pat. Nos. 4,990,607, 4,797,359, 5,521,084, and 5,447,858. Overexpression of hsp-70 has been accomplished in transgenic mice. Plumeer et al., (1995) J. Clin. Inv. 95:1854–1860. See also Yost and Lindquist, (1986) Cell 45:185–193, Yost and Lindquist, (1988) Science 242:1544–1548, Garbe et al. (1986) PNAS, 83:1812–1816, Blackman et al. (1986) J. Mol. Biol. 188:499–515, Bond et al. (1986) Mol. Cell. Biol. 12:4602–4610, Kay et al. (1987) Nucl. Acids Res. 15:3723–3741, Bond, (1988) EMBO J. 7:3509–3518. The published sequences of these promoters are hereby incorporated by reference.
The inducible promoter systems which have been used to control the expression of proteins typically have one or more of the following limitations: they are restricted to a relatively narrow range of host cells, or are only partially inducible, or are derived from organisms, such as tumor viruses, which are inherently dangerous.
More importantly, their use does not allow for finely tuned localized expression of exogenous genes in transformed cells. “Among the design hurdles for all vectors are . . . to enable the transferred gene to be regulated”. Crystal (1995), “Transfer of genes to humans: Early lessons and obstacles to success.”